Banking of multipotent amniotic fetal stem cells

ABSTRACT

Stem cells, including those designated as multipotent amniotic fluid stem cells (MAFSC) cells are found in the amniotic fluid of mammals, including humans. MAFSCs are fetal, multipotent stem cells that can be used for any desired stem cell utility, including treatment of individuals in need of tissue replacement or gene therapy. Methods of banking MAFSCs derived from the amniotic fluid cells of pregnant individuals are disclosed. Amniotic fluid-derived cells are banked for the purpose of access to transplantation antigen-compatible or syngeneic multipotent stem cells.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119 to U.S.Provisional Application No. 60/495,513, filed Aug. 14, 2003, and U.S.Provisional Application No. 60/495,437, filed Aug. 14, 2003, thedisclosures of which are incorporated by reference herein in theirentireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of stem cell research. Specifically,the invention relates to the preservation and banking of amnioticfluid-derived cells, or multipotent amniotic fluid-derived stem cells ofindividuals. The cryopreserved amniotic fluid-derived cells can bestored indefinitely. Thawed cells can be used to grow stem cell lineswhich can create differentiated cells, such as specific cell types ortissue types. These differentiated cells are capable of beingtransplanted into the individual or into unrelated matching individualsif needed.

2. Description of the Related Art

Stem cells can give rise to many types of differentiated cells, and thusmay be useful to treat many types of diseases. Stem cells have theability to divide for indefinite periods in culture and to give rise tospecialized cells. There are several types of stem cells, such asembryonic stem cells, which are undifferentiated cells from the embryo,and adult stem cells, which are undifferentiated cells derived fromvarious mature tissues.

Embryonic stem cells have the potential to become a wide variety ofspecialized cell types. This ability of an embryonic stem cell to becomea variety of cell types is termed “pluripotent.” Embryonic stem cellscan be differentiated into a host of cell types and tissue types whichcan be used for basic research, drug discovery, treatment and preventionof diseases. For example, U.S. Pat. No. 6,506,574 to Rambhatla, which isincorporated by reference herein in its entirety, discloses methods ofdifferentiating embryonic stem cell cultures into hepatocyte lineagecells. Other methods for the preparation of embryonic stem cells aredisclosed, for example, in U.S. Pat. No. 6,200,806 to Thomson; U.S. Pat.No. 5,670,372 to Hogan, and U.S. Pat. No. 6,432,711 to Dinsmore, each ofwhich is incorporated by reference herein in its entirety.

Human Embryonic Stem cells (hES) are derived from the inner cell mass ofthe blastocyst, the earliest stage of embryonic development of thefertilized egg. The blastocyst is a preimplantation stage of the embryo,a stage before the embryo would implant in the uterine wall. Whencultured on an inactivated feeder layer of cells according to conditionsdescribed by Thompson and colleagues (Thomson, et al., (1995) Proc.Natl. Acad. Sci. U.S.A. 92:7844-7848; Thomson, et al. (1998) Science282:1145-1147; Marshall, et al., (2001) Methods Mol. Biol. 158:11-18,each of which is incorporated by reference herein in its entirety theinner layer cells of the blastocyst can be grown in vitro indefinitelyin an undifferentiated state. Properly propagated hES cells haveunlimited potential to double while maintaining their capacity ofdifferentiating into the three layers of the embryo, Ectoderm (Ec),Mesoderm (me) and Endoderm (En); they are pluripotent. When grown aspluripotent hES, the cells maintain a euploid karyotype and are notprone to senescence. hES cells have been differentiated in vitro intoskin and brain (Ec), heart, muscle, kidney and blood (Me), and intopancreatic, thyroid and lung cells (En) (Fraichard, et al., (1995) JCell Sci. 108:3181-3188; Itskovitz-Eldor, et al., (2000). Mol. Med.6:88-95; Lee, et al., (2000) Nat. Biotechnol. 18:675-679; Liu, et al.,(2000) Proc. Natl. Acad. Sci. U.S.A. 97:6126-6131; Lumelsky, et al.,(2001) Science 292:1389-1394; Maltsev, et al., (1993). Mech. Dev.44:41-50; Odorico, et al., (2001) Stem Cells 19:193-204. Potocnik, etal., EMBO. J. 13:5274-5283; Reubinoff, et al., (2000) Nat. Biotechnol.18:399-404; Schuldiner, et al., (2001) Proc. Natl. Acad. Sci. USA97:1997:11307-11312; Kim, et al., (2002) Nature 418:50-56; Wichterle, etal., (2002) Cell 110:385-397), each of which is incorporated byreference herein in its entirety.

Human embyronic stem cells display a distinct group of cell surfaceantigens, SSEA-3, SSEA-4, TRA-2-54 (alkaline phosphatase), TRA-1-60 andTRA-1-81, in addition to expressing specific transcription factorsOCT-4, NANOG, SOX-2, FGF-4 and REX-1 (Henderson, et al., (2002) StemCells 20:329-337; Draper, et al., (2002). J. Anat. 200:249-258; Mitsuiet al., (2003) Cell 113:631-642; Chambers et al., (2003) Cell113:643-655), each of which is incorporated by reference herein in itsentirety.

Additionally, hES cells (i) are capable of symmetrical division in vitrowithout differentiating; (ii) can integrate into all fetal tissuesduring in vivo development; (iii) are capable of colonizing the germline and give rise to egg or sperm cells; (iv) develop intoteratocarcinomas in immunologically impaired adult mice—another measureof pluripotency, and lack the G1 checkpoint in the cell cycle likesomatic cells but spend most of their time in S phase.

Stem cells can also be derived from nonembryonic sources. For example,an additional class of human stem cells are the mesenchymal or adultstem cells (MSC). Adult stem cells are undifferentiated, like embryonicstem cells, but are present in differentiated tissues. Adult stem cellsare capable of differentiation into the cell types from the tissue thatthe adult stem cell originated. Adult stem cells (MSC) have been derivedfrom the nervous system (McKay, R. (1997) Science 276:66-71.Shihabuddin, et al., (1999) Mol. Med. Today 5:474-480), bone marrow(Pittenger, et al., (1999) Science 284:143-147; Pittenger, M. F. andMarshak, D. R. (2001). In: Mesenchymal stem cells of human adult bonemarrow. Marshak, D. R., Gardner, D. K., and Gottlieb, D. eds. (ColdSpring Harbor, N.Y.: Cold Spring Harbor Laboratory Press) 349-374);adipose tissue (Gronthos, et al., (2001) J. Cell. Physiol. 189:54-63),dermis (Toma, et al., (2001) Nature cell Biol. 3:778-784) and pancreasand liver (Deutsch, et al., (2001) Development 128:871-881), each ofwhich is incorporated by reference herein in its entirety.

The political, moral and ethical issues around hES cells, as well as theperceived difficulties of expanding undifferentiated adult stem cells inculture, while maintaining a genetically normal genome, are majorbarriers in the development of human cell replacement therapy. Further,what is needed in the art is a method of isolating, storing, banking,and retrieving novel sources of multipotent or pluripotent human stemcells so that they can be revived and utilized at a later date.

SUMMARY OF THE INVENTION

Embodiments of the invention include a cell bank system havingpreserved, viable samples from many individuals, where the samplescontain amniotic fluid-derived cells, and a database allowing forspecific identification and retrieval of individual samples. Thedatabase may include information allowing association of an individualsample with the sample donor, and the sample may be retrieved by thedonor. The samples may contain amniotic fluid, and may containmultipotent or pluripotent stem cells. The amniotic fluid-derived cellscomprise pluripotent stem cells, or can be multipotent stem cells. Forexample, the amniotic fluid-derived cells can be multipotent stem cellscharacterized by a) the ability to grow in continuous culture for atleast 60 generations, and b) the presence of at least one, or two, orthree, or four, or five, or all of the markers selected from the groupconsisting of: SSEA3, SSEA4, Tra1-60, Tra1-81, Tra2-54, and Oct-4. Thestem cells can further express at least one marker selected from thegroup consisting of: HLA Class I, CD13, CD44, CD49b, and CD105. Theamniotic fluid-derived cells can be pluripotent stem cells characterizedby a) the ability to grow in continuous culture for at least 60generations, and b) the presence of at least one, or two, or three, orfour, or five, or all of the markers selected from the group consistingof: SSEA3, SSEA4, Tra1-60, Tra1-81, Tra2-54, and Oct-4. The stem cellscan further express at least one marker selected from the groupconsisting of: HLA Class I, CD13, CD44, CD49b, and CD105.

Additional embodiments of the invention include methods for banking stemcells by obtaining viable samples containing amniotic-fluid-derived stemcells, which were derived from the amniotic fluid of individual fetuses,then preserving and storing the samples in a way that allows viabilityof at least some of the stem cells, storing data relating to theidentity of the individual samples; and allowing the retrieval ofindividual samples by the individual from whom the sample was obtained.The samples may be cryopreserved, and may contain MAFCS, amniotic fluid,multopotent and/or pluripotent stem cells.

Further embodiments of the invention include methods for banking stemcells by obtaining viable samples containing amniotic-fluid-derived stemcells derived from the amniotic fluid of individual fetuses, preservingand storing the samples in a way that preserves viability of at leastsome of the stem cells, storing data relating to tissue compatibilitycharacteristics of individual samples; and providing for retrieval of anindividual sample by an individual sharing tissue compatibilitycharacteristics of the sample. Such samples may be cryopreserved, andmay contain MAFCS, amniotic fluid, multopotent and/or pluripotent stemcells.

Yet further embodiments of the invention include collections havingnumerous viable stem cell samples derived from amniotic fluid ofdifferent fetuses. Such samples may be cryopreserved, and may containMAFCS, amniotic fluid, multopotent and/or pluripotent stem cells.

Additional embodiments of the invention include methods of preparingamniotic fluid-derived cells for a potential future use, by obtainingand cryopreserving amniotic fluid containing live amniotic stem cells.The cryopreserving step may be performed in a medium which containsDMSO, preferably at 1% to 80%, and more preferably 10% to 40% (V/V)DMSO. Amniotic fluid may also be present, preferably at about 15% to 35%(V/V).

Further embodiments of the invention include methods for preparingamniotic fluid derived stem cells or multipotent amniotic fluid derivedstem cells (MAFSC) for a potential future use by obtaining amnioticfluid containing live cells, then culturing MAFSC cells isolated fromsaid amniotic fluid, then cryopreserving the MAFSC cells. Thecryopreserving step may be performed in a medium which contains DMSO,preferably at 1% to 80%, and more preferably 10% to 40% (V/V) DMSO.Amniotic fluid may also be present, preferably at about 15% to 35%(V/V).

Additional embodiments include storage banks of multiple samples ofamniotic fluid-derived cells or MAFSCs taken from multiple individuals,where the samples are preserved in such a way as to be viable uponrecovery. The cells may be cryopreserved in a medium having DMSO. Atleast some of the samples can be differentiable human stem cells, whichmay be multipotent or pluripotent.

Additional embodiments include data storage media containing a databasehaving a first data item associated with each of many preservedamniotic-fluid-derived stem cell samples, containing locationinformation for the samples; and a second data item associated with eachsample containing identifying information for the sample. The seconddata item can include, for example, the identity of a fetus from whomthe sample was obtained, tissue compatibility information, and/orhistocompatibility information for the stem cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

A newly discovered source of human stem cells is described herein and inco-pending application Ser. No. 60/495,437, which is incorporated byreference herein in its entirety.

The cells, coined Multipotent Amniotic Fetal Stem Cells (MAFSC), areimmortal in culture, maintain euploidy for >1 year in culture, sharemarkers with human ES cells, and are capable of differentiating into allthree germ layers of the developing embryo, Endoderm, Mesoderm andEctoderm. These human stem cells are found in the amnion harvestedduring the first trimester of human pregnancies.

Both fresh amniotic fluid derived cells, and the cultured MAFSC cellsderived from them, may be stored indefinitely. When a need arises, analiquot of the cells can be thawed, cultured, and used as needed. Thelong term storage of amniotic fluid derived cells allows an individualto have a supply of stem cells taken while the individual is still inthe womb, to be stored in such a way as to provide a supply of cells foremergency or other uses throughout the individual's lifetime. The longterm storage or “banking” of amniotic fluid-derived cells or culturedMAFSCs is disclosed herein.

While amniotic fluid contains multiple morphologically-distinguishablecell types, the majority of the cells are prone to senescence and arelost from cultures grown under MAFSC culture conditions. More than 80%of amniotic fluid harvests from normal 16-18 week pregnancies give riseto continuous MAFSC lines. The MAFSCs may be harvested from anmnioticfluid from pregnant females at any stage in the gestation period.

MAFSC are of fetal origin, and have a normal diploid karyotype.Additionally, MAFSC cells are devoid of tumorgenic properties: unlikehEC cells, human MAFSC cells do not grow into teratocarcinomas wheninjected into SCID mice. This property may be especially useful in usingMAFSC cells or their derivatives for human gene therapy purposes.

The term “stem cell” refers to any cells that have the ability to dividefor indefinite periods of time and to give rise to specialized cells.The term “long term stem cells” refers more specifically to those stemcells that are capable of self-renewal over indefinite periods of time.

The MAFSC cells of the invention have been shown to be multipotent, asseveral main cell types have been derived from them. As used herein, theterm “multipotent” refers to the ability of MAFSC to differentiate intoseveral main cell types. The MAFSC cells may also be propagated underspecific conditions to become “pluripotent.” The term “pluripotent stemcells” describes stem cells that are capable of differentiating into anytype of body cell, when cultured under conditions that give rise to theparticular cell type.

The MAFSCs may be isolated as described, for example, in Example 1.Briefly, the sample of amniotic fluid (AF) can be was removed from apregnant female at any time during thee gestation period. Cells to becultured are then removed from the amniotic fluid, preferably bycentrifugation or filtration. The cells can then be plated on medium asdisclosed in Example 1, or other suitable growth medium.

Typically, the cells are grown in a nutrient medium such as the mediumshown in Example 1. Expansion of the undifferentiated amnioticfluid-derived cells can be achieved by culturing the cells in definedmedia containing low amounts of serum or no serum at all, using, forexample, recombinant growth promoting factors. The term“undifferentiated” refers to cells that have not become specialized celltypes. A “nutrient medium” is a medium for culturing cells containingnutrients that promote proliferation. The nutrient medium may containany of the following in an appropriate combination: isotonic saline,buffer, amino acids, antibiotics, serum or serum replacement, andexogenously added factors.

The MAFSCs are preferably isolated from humans. However, the MAFSCs maybe isolated in a similar manner from other species. Examples of speciesthat may be used to derive the MAFSCs include but are not limited tomammals, humans, primates, dogs, cats, goats, elephants, endangeredspecies, cattle, horses, pigs, mice, rabbits, and the like.

The amniotic fluid-derived cells and MAFSC can be recognized by theirspecific cell surface proteins or by the presence of specific cellularproteins. Typically, specific cell types have specific cell surfaceproteins. These surface proteins can be used as “markers” to determineor confirm specific cell types. Typically, these surface markers can bevisualized using antibody-based technology or other detection methods.The surface markers of the isolated MAFSC cells derived fromindependently-harvested amniotic fluid samples were tested for a rangeof cell surface and other markers, using monoclonal antibodies and FACSanalysis (see Examples 2 and 3, and Table 1). These cells arecharacterized by the following cell surface markers: SSEA3, SSEA4,Tra1-60, Tra1-81, Tra2-54 but are distinguished from mouse ES cells inthat these cells do not express the cell surface marker SSEA1.Additionally, MAFSC express the stem cell transcription factor Oct-4.

Storage Bank of Amniotic Fluid-Derived Cells

The anmiotic fluid-derived cells can be stored or “banked” in a mannerthat allows the cells to be revived as needed in the future. An aliquotof the undifferentiated cells can be removed at any time, to be growninto cultures of many undifferentiated cells and then differentiatedinto a particular cell type or tissue type, and may then be used totreat a disease or to replace malfunctioning tissues in a patient. Sincethe cells are harvested from the amniotic fluid, the cells can be storedso that an individual can have access to his or her own undifferentiatedcells for an entire lifetime. Alternatively, the cells can be used byindividuals other than the original donor.

In addition, although the principal exemplary disclosure of the presentapplication relates to amniotic stem cells, the cell banking andretrieval disclosure herein is similarly applicable to any stem celltypes, including those derived from embryos, placenta, umbilicus,infants, children, and adults.

In one embodiment of the present invention, a stem cell bank is providedfor storing amniotic fluid-derived cell samples. In additionalembodiments of the present invention, methods for administering such astem cell bank are provided. U.S. Published Patent Application No.20030215942, which is incorporated by reference herein in its entirety,provides an example of a stem cell bank system.

Using methods such as those described above and in Examples 5 and 6,below, the isolation and in vitro propagation of stem cell samples fromamniocentesis harvests and their cryopreservation facilitates theestablishment of a “bank” of transplantable human stem cells. The methoddescribed herein allows viable stem cells of any individual to beobtained from the amniotic fluid (for example, from an amniocentesisprocedure) and be available for use at any time in the future. Anynumber of individuals may have cells stored in this manner. Because itis possible to store smaller aliquots of AF or MASFCs, the bankingprocedure could take up a relatively small space. Therefore, the cellsof many individuals could be stored or “banked” on a short term or longterm basis, with relatively little expense.

In some embodiments of the present invention, a portion of the sample ismade available for testing, either before or after processing andstorage.

Cryopreservation Methods

The fresh amniotic fluid-derived cells, or the cultured MAFSC cells maybe preserved so that the cells can be revived on demand. A preferredmethod is cryopreservation. One example of a suitable cryopreservationmethod is shown in Example 4. Typically, the surrounding fluid forpreservation contains amniotic fluid. The use of amniotic fluid as partof the cryopreservation medium, rather than the use of other types ofmedia or serum, allows for the cells to remain in the preferredundifferentiated form. Exposure of primary amniotic fluid (AF) cells orMAFSC cells to serum may cause the cells to become more susceptible tocontrolled differentiation, which may make them less suitable for futuremultipotent or pluripotent uses, once they are removed from storage.Preferably, the cryopreservation medium contains between about 10% and50% amniotic fluid. More preferably, the cryopreservation mediumcontains between about 20% and 30% amniotic fluid. Most preferably, thecryopreservation medium contains about 24% to about 27% amniotic fluid.Preferably, the amniotic fluid is filtered. Most preferably, thefiltration occurs through a 0.1 μm filter.

Another component of the cryopreservation medium is DMSO(dimethylsulfoxide). Preferably, DMSO is present at approximately 1% to80% (V/V). More preferably, DMSO is present at approximately 5% to 30%(V/V). Most preferably, DMSO is present at approximately 8% to 12% DMSO.

The AF cells or MAFSCs can be stored indefinitely under liquid nitrogen.The cells can be kept for >100 years without the incidence of anydamage: they can then be thawed, grown and differentiated as required.The cells are preferably frozen in the above described medium at acontrolled rate. Preferably, this rate is from about 0.1° C./min toabout 10° C./min. More preferably, the freezing rate is from about 0.2,0.3, 0.4° C./min to about 4, 6, 8, or 9° C./min. Most preferably, thefreezing rate is from about 0.5° C./min to about 2° C./min.

Frozen cells are then stored under liquid nitrogen until needed. Thecells may be stored indefinitely, once frozen. Care should be taken toprevent the possibility of accidental thawing or warming of the frozencells at any time during their storage period. In some embodiments ofthe invention, the cells may be preserved by methods other thancryopreservation.

Typing of Amniotic Fluid-Derived Cell Samples

In some embodiments of this invention, the amniotic fluid-derived cellscan be further classified according to certain identifying features,such as by HLA typing, either before or after processing and storage.The term “type or “typing” refers to any characteristics of an amnioticfluid-derived cell sample that may be relevant for any possible use ofthe sample. Determination of which tests are relevant and how to performthem is entirely conventional and will change with technologicaldevelopments. Typing also includes any method that identifies a stemcell product in such a way that the stem cell sample may be matched to acertain individual. For the purposes of this invention, matchingindicates that the stem cell sample is suitable for transplantation intoa specific individual.

The type information may include, for example, genotype or phenotypeinformation. Genotype information may refer to a specific geneticcomposition of a specific individual organism, for example, whether anindividual organism has one or more specific genetic variants up to allthe variations in that individual's genome, for example, whether theindividual is a carrier of genetic variations that influence disease orthe HLA type of that individual. Phenotype information may include anyobservable or measurable parameter.

In some embodiments of the invention, the amniotic fluid-derived cellsmay be typed using HLA typing methods. For example, stem cells can betyped using the high-throughput HLA typing-methods described in U.S.Pat. No. 6,670,124, which is incorporated by reference herein in itsentirety. A high throughput HLA typing method may include obtaining abiological sample containing template nucleic acid from a subject,amplifying the template nucleic acid with labeled HLA allele-specificprimers, hybridizing the amplification products with immobilized HLAlocus-specific capture oligonucleotides and using detection methods todetermine the HLA genotype of the subject.

Other typing methods may be used. One typing method for HLAidentification purposes is restriction fragment length polymorphismanalysis. Restriction fragment length polymorphism analysis relies uponthe strong linkage between allele-specific nucleotide sequences withinthe exons that encode functionally significant HLA class II epitopes.Another method, PCR-SSO, relies upon the hybridization of PCR amplifiedproducts with sequence-specific oligonucleotide probes to distinguishbetween HLA alleles (Tiercy et al., 1990, Blood Review 4: 9-15, Saiki etal., 1989, Proc. Natl. Acad. Sci., U.S.A. 86: 6230-6234; Erlich et al.(1991) Eur. J. Immunogenet. 18(1-2): 3355; Kawasaki et al. (1993)Methods Enzymol. 218:369-381), each of which is incorporated byreference herein in its entirety.

Another molecular typing method that can be used in the presentinvention, PCR-SSP, uses sequence specific primer amplification (Olerupand Zetterquist (1992) Tissue Antigens 39: 225-235, which isincorporated by reference herein in its entirety). TheSSCP-Single-Stranded Conformational Polymorphism method may also beused. These and other standard techniques for HLA typing are known inthe art, e.g., DNA typing or serological and cellular typing (Terasakiet al., 1964, Nature, 204:998, which is incorporated by reference hereinin its entirety).

In some embodiments of the invention, in order to allow for theavailability of cells which can be used for any individual, even ifthose who do not have stored amniotic fluid-derived cell samples, manyamniotic fluid-derived cell samples can be banked that possess a rangeof genetic characteristics and that display a range of antigens to allowfor sufficient matching of HLA specificities for the use by anypotential recipient.

Organizing the Amniotic Fluid-Derived Cell Samples

This invention also provides methods of recording the amnioticfluid-derived cell samples so that when a stem cell sample needs to belocated, it can be easily retrieved. Any indexing and retrieval systemcan be used to fulfill this purpose. Any suitable type of storage systemcan be used so that the stem cells can be stored. The amnioticfluid-derived cell samples can be designed to store individual samples,or can be designed to store hundreds, thousands and even millions ofdifferent amniotic fluid-derived cell samples.

The stored amniotic fluid-derived cell samples can be indexed forreliable and accurate retreival. For example, each sample can be markedwith alphanumeric codes, bar codes, or any other method or combinationsthereof. There may also be an accessible and readable listing ofinformation enabling identification of each stem cell sample and itslocation in the bank and enabling identification of the source and/ortype of stem cell sample, which is outside of the bank. This indexingsystem can be managed in any way known in the art, e.g., manually ornon-manually, e.g. a computer and conventional software can be used.

In some embodiments of the invention, the amniotic fluid-derived cellsamples are organized using an indexing system so that the sample willbe available for the donor's use whenever needed. In other embodimentsof the invention, the amniotic fluid-derived cell samples can beutilized by individuals other than the original donor. Once recordedinto the indexing system, the amniotic fluid-derived cell sample can bemade available for matching purposes, e.g., a matching program willidentify an individual with matching type information and the individualwill have the option of being provided the matching stem cell sample.

The storage banking system can comprise a system for storing a pluralityof records associated with a plurality of individuals and a plurality ofamniotic fluid-derived cell samples. Each record may contain typeinformation, genotypic information or phenotypic information associatedwith the stem cell samples or specific individuals. In a specificembodiment, the system will include a cross-match table that matchestypes of the stem cell samples with types of individuals who with toreceive a stem cell sample.

In a particular embodiment, the database system stores information foreach stem cell sample in the bank. Certain information is stored inassociation with each sample. The information may be associated with aparticular donor, for example, an identification of the donor and thedonor's medical history. Alternatively, a stem cell sample may beanonymous and not associated with a specific donor. Alternatively, oradditionally, the information may be sample type information. Forexample, the information might include the volume of the stem cellsample or the total nucleated cells count in the product. The storedinformation may also include match and typing information. For example,each stem cell sample may be HLA typed and the HLA type information maybe stored in association with each sample. The information stored mayalso be availability information. The information stored with eachsample is searchable and identifies the sample in such a way that it canbe located and supplied to the client immediately.

Accordingly, Embodiments of the invention utilize computer-based systemsthat contain information such as the donor, date of submission, type ofcells submitted, types of cell surface markers present, geneticinformation relating to the donor, or other pertinent information, andstorage details such as maintenance records and the location of thestored samples, and other useful information.

The term “a computer-based system” refers to the hardware, software, andany database used to store, search, and retrieve information about thestored cells. The computer-based system preferably includes the storagemedia described above, and a processor for accessing and manipulatingthe data. The hardware of the computer-based systems of this embodimentcomprise a central processing unit (CPU) and a database. A skilledartisan can readily appreciate that any one of the currently availablecomputer-based systems are suitable.

In one particular embodiment, the computer system includes a processorconnected to a bus that is connected to a main memory (preferablyimplemented as RAM) and a variety of secondary storage devices, such asa hard drive and removable medium storage device. The removable mediumstorage device can represent, for example, a floppy disk drive, a DVDdrive, an optical disk drive, a compact disk drive, a magnetic tapedrive, etc. A removable storage medium, such as a floppy disk, a compactdisk, a magnetic tape, etc. containing control logic and/or datarecorded therein can be inserted into the removable storage device. Thecomputer system includes appropriate software for reading the controllogic and/or the data from the removable medium storage device onceinserted in the removable medium storage device. Information relating tothe amniotic fluid-derived cells can be stored in a well known manner inthe main memory, any of the secondary storage devices, and/or aremovable storage medium. Software for accessing and processing thesesequences (such as search tools, compare tools, etc.) reside in mainmemory during execution.

As used herein, “a database” refers to memory that can store any usefulinformation relating to the amniotic fluid-derived cell collections andthe donors. Additionally, a “database” refers to a memory accesscomponent that can access manufactures having recorded thereoninformation relating to the amniotic fluid-derived cell collections.

The data relating to the stored amniotic fluid-derived cells can bestored and manipulated in a variety of data processor programs in avariety of formats. For example, the data can be stored as text in aword processing file, such as Microsoft WORD or WORDPERFECT, an ASCIIfile, a html file, or a pdf file in a variety of database programsfamiliar to those of skill in the art, such as DB2, SYBASE, or ORACLE.

A “search program” refers to one or more programs that are implementedon the computer-based system to search for details or compareinformation relating to the cryopreserved samples within a database. A“retrieval program” refers to one or more programs that can beimplemented on the computer-based system to identify parameters ofinterest in the database. For example, a retrieval program can be usedto find samples that fit a particular profile, samples having specificmarkers or DNA sequences, or to find the location of samplescorresponding to particular individuals.

Storage Facility

The amniotic fluid-derived cell samples of the invention may betransported to and from a cell storage facility, interim facility orprocessing area by methods known in the art. Storage of the amnioticfluid-derived cell samples may be short term or long term. In someembodiments, the stored cells may be cryogenically preserved but anystorage method suitable for long term storage may be used, such as, forexample, the addition of amino acids, inosine, adenine, or othercompounds to the cells. Any storage method may be used in this inventionproviding that the stored product retain viability for the therapeuticor other purposes.

There is no upper limit on the number of amniotic fluid-derived cellsamples that can be stored in one cell bank. In one embodiment, hundredsof stem cell products from different individuals will be stored at onebank or storage facility. In another embodiment, up to millions ofproducts may be stored in one storage facility. A single storagefacility may be used to store amniotic fluid-derived cell samples, ormultiple storage facilities may be used.

In some embodiments of the present invention, the storage facility mayhave a means for any method of organizing and indexing the stored cellsamples, such as, for example, automated robotic retrieval mechanismsand cell sample manipulation mechanisms. The facility may includemicromanipulation devices for processing such amniotic fluid-derivedcell samples. Known conventional technologies can be used for efficientstorage and retrieval of the amniotic fluid-derived cell samples.Exemplary technologies include but are not limited to Machine Vision,Robotics, Automated Guided Vehicle System, Automated Storage andRetrieval Systems, Computer Integrated Manufacturing, Computer AidedProcess Planning, Statistical Process Control, and the like. Lesssophisticated storage facilities may be used as well, such as, forexample, large areas maintained at appropriate temperatures havingnumerous racks on which are indexed and stored the amnioticfluid-derived cell samples of the invention.

Potential Recipients of the Amniotic Fluid-Derived Cells

The type information or other information associated with the individualin need of a amniotic fluid-derived cell sample may be recorded into asystem that can be used to identify an appropriate matching stem cellproduct, such as, for example, a database system, an indexing system,and the like. Once recorded in the system, a match can be made betweenthe type of the individual and a donor amniotic fluid-derived cellsample. In preferred embodiments, the donor sample is from the sameindividual as the individual in need of the sample. However, similar butnot identical donor/recipient matches can also be used. The matchingamniotic fluid-derived cell sample is available for the individualpossessing the matching type identifier. In one embodiment of thisinvention, the individual's identification information is stored inconnection with the cell sample. In some embodiments, the matchingprocess occurs around the time of harvesting the sample, or can occur atany time during processing, storage, or when a need arises. Accordingly,in some embodiments of the invention, the matching process occurs beforethe individual is in actual need of the amniotic fluid-derived cellsample.

When the amniotic fluid-derived cell sample is needed by an individual,it may be retrieved and made available for research, transplantation orother purposes within minutes, if desired. The stem cell sample may alsobe further processed to prepare it for transplantation or other needs.

Thawing the Banked Cells and Use of the Thawed Cells

When the cells are to be used, they can be thawed under controlledconditions. An example of one suitable method is shown in Example 8.Preferably, the thawing is performed at about 37° C. A water bath set at37° C. may be used for this purpose.

The thawed samples can then be tested for viability and growthcharacteristics. Typically, over 99% viability is attained. The cellscan be grown in any suitable medium, once the DMSO is diluted to lessthan about 1% of the cell culture volume. The growth properties,viability, karyotype and differentiation ability of frozen and thawedcells were found to be identical to fresh AF cells and to MAFSC cellsupon freezing, respectively.

It was found that the previously stored, thawed cells could be grown anddifferentiated as if they had not been frozen. Therefore, once thawed,the cells can be used for creating cultures of undifferentiated cells,or for creating differentiated cell types or tissue types as disclosedherein and in co-pending U.S. patent provisional application Ser. No.60/495,437, which is incorporated by reference herein in its entirety.

The thawed MAFSCs may be grown in an undifferentiated state for as longas desired, and can then be cultured under certain conditions to allowprogression to a differentiated state. By “differentiation” is meant theprocess whereby an unspecialized cell acquires the features of aspecialized cell such as a heart, liver, muscle, pancreas or other organor tissue cell. The MAFSCs, when cultured under certain conditions, havethe ability to differentiate in a regulated manner into three or moresubphenotypes. Once sufficient cellular mass is achieved, cells can bedifferentiated into endodermal, mesodermal and ectodermal derivedtissues in vitro and in vivo. This planned, specialized differentiationfrom undifferentiated cells towards a specific cell type or tissue typeis termed “directed differentiation.” Examples of such cell types thatmay be prepared from MAFSCs using directed differentiation include butare not limited to fat cells, cardiac muscle cells, epithelial cells,liver cells, brain cells, blood cells, neurons, or glial cells.

General methods relating to stem cell differentiation techniques thatmay be useful for differentiating the MAFSCs of this invention can befound in general texts such as: Teratocarcinomas and embryonic stemcells: A practical approach (E. J. Robertson, ed., IRL Press Ltd. 1987);Guide to Techniques in Mouse Development (P. M. Wasserman et al. eds.,Academic Press 1993); Embryonic Stem Cell Differentiation in vitro (M.V. Wiles, Meth. Enzymol. 225:900, 1993); Properties and uses ofEmbryonic Stem Cells: Prospects for Application to Human Biology andGene Therapy (P. D. Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998);and in Stem cell biology (L. M. Reid, Curr. Opinion Cell Biol. 2:121,1990), each of which is incorporated by reference herein in itsentirety.

Differentiation agents, maturation agents, or maturation factors may beuseful to allow progression to certain cell types. Examples ofdifferentiation agents, that may be used include but are not limited toagents, such as N-butyrate, which are useful for differentiatingembryonic stem cells to liver cells are described in U.S. Pat. No.6,506,574, to Rambhatla et al. Optionally, maturation agents, ormaturation factors, such as, for example, growth factors, peptidehormones, cytokines, ligand receptor complexes, corticosteroids, andeven organic solvents like DMSO have been found to effectdifferentiation of embryonic stem cells (U.S. Pat. No. 6,506,574, whichis incorporated by reference herein in its entirety.

Treatment of Individuals Using Amniotic Fluid-Derived Cells that havebeen Thawed from a Cell Bank

The isolated amniotic fluid-derived cells or their derivatives may beused to treat diseases in humans or animals. As used herein the term“treat” or “treatment” refer to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to prevent,slow down (lessen), or reverse an undesired physiological change ordisorder. The term “treat” also refers to the characterization of thetype or severity of disease which may have ramifications for futureprognosis, or need for specific treatments. For purposes of thisinvention, beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

To treat a human or animal in need of treatment, the amnioticfluid-derived cells can be either regenerated into segments of a desiredtissue, then transplanted into the patient, or can be regenerated into awhole tissue that will be used to replace the failing tissue, or can beinjected into a tissue of interest as whole cells, where they willregenerate at the injected location.

The banked amniotic fluid-derived cells may be used in the individual'spostnatal life for purposes such as regenerative medical attention (asshown in Examples 8 and 9) or for cosmetic purposes. The banked cellswill be readily available to prepare replacement tissue or cells asneeded. Additionally, the amniotic fluid-derived cells and tissuesderived from amniotic fluid-derived cells can also be shared withindividuals of similar, but not necessarily identical genetic make-up. Adirected search of the database information can be used to find samplesof similar but not necessarily identical genetic make-up in situationswhere individuals of similar genetic background are not known or are notavailable.

It may be possible to replace any type of failing tissue with amnioticfluid-derived cells. Accordingly, amniotic fluid-derived cells that havebeen retrieved from a cell bank system may be differentiated intotissues such as liver, endocrine tissues, lung, blood cells, neuronal orastroglial cells, or others, which may then be used for transplantationto cure or treat diseases. Examples of tissues which may be (at leastpartially) replaced include, but are not limited to pancreatic tissue orcells, lung tissue, heart tissue, ocular tissue, nerve tissue, braintissue, muscle tissue, skin, or others.

Examples of diseases that may be treated with amniotic fluid-derivedcell-derived cells or tissues include but are not limited to cirrhosisof the liver, pancreatitis, diabetes, Parkinson's disease, spinal cordinjury, stroke, burns, heart disease, certain types of cancer,osteoarthritis, rheumatoid arthritis, leukemia, lymphoma, genetic blooddisorders, Examples of diseases that can be treated with amnioticfluid-derived cell-derived stem cells include but are not limited toAcute Lymphoblastic Leukemia, Acute Myelogenous Leukemia, AcuteBiphenotypic Leukemia, and Acute Undifferentiated Leukemia; ChronicMyelogenous Leukemia, Chronic Lymphocytic Leukemia, Juvenile ChronicMyelogenous Leukemia, Juvenile Myelomonocytic Leukemia, RefractoryAnemia, Refractory Anemia with Ringed Sideroblasts, Refractory Anemiawith Excess Blasts, Refractory Anemia with Excess Blasts inTransformation, Chronic Myelomonocytic Leukemia, Aplastic Anemia,Fanconi Anemia, Paroxysmal Nocturnal Hemoglobinuria, Pure Red CellAplasia, Acute Myelofibrosis, Agnogenic Myeloid Metaplasia,myelofibrosis, Polycythemia Vera, Essential Thrombocythemia,Non-Hodgkin's Lymphoma, Hodgkin's Disease, Chediak-Higashi Syndrome,Chronic Granulomatous Disease, Neutrophil Actin Deficiency, ReticularDysgenesis, Mucopolysaccharidoses, Hurler's Syndrome, Scheie Syndrome,Hunter's Syndrome, Sanfilippo Syndrome, Morquio Syndrome, Maroteaux-LamySyndrome, Sly Syndrome, Beta-Glucuronidase Deficiency,Adrenoleukodystrophy, Mucolipidosis II, Krabbe Disease, Gaucher'sDisease, Niemann-Pick Disease, Wolman Disease, MetachromaticLeukodystrophy, Familial Erythrophagocytic Lymphohistiocytosis,Histiocytosis-X, Hemophagocytosis, Inherited Erythrocyte Abnormalities,Beta Thalassemia Major, Sickle Cell Disease, Inherited Immune SystemDisorders, Ataxia-Telangiectasia, Kostmann Syndrome, Leukocyte AdhesionDeficiency, DiGeorge Syndrome, Bare Lymphocyte Syndrome, Omenn'sSyndrome, Severe Combined Immunodeficiency, Common VariableImmunodeficiency, Wiskott-Aldrich Syndrome, X-Linked LymphoproliferativeDisorder, Other Inherited Disorders, Lesch-Nyhan Syndrome,Cartilage-Hair Hypoplasia, Glanzmann Thrombasthenia, Osteopetrosis,Inherited Platelet Abnormalities, Amegakaryocytosis, CongenitalThrombocytopenia, Plasma Cell Disorders, Multiple Myeloma, Plasma CellLeukemia, Waldenstrom's Macroglobulinemia, Breast Cancer, Ewing Sarcoma,Neuroblastoma, Renal Cell Carcinoma, brain disorders such as Alzheimer'sdisease, and the like (see, for example, hypertext transfer protocol(http) on the world wide web at the following link:marrow.org/index.html, which is incorporated by reference herein in itsentirety).

Genetic Modification of MAFSCs before or after Banking

MAFSCs or MAFSC-derived cells may also be genetically modified bytransduction with any suitable gene of interest, as shown in Example 10.General techniques useful to genetically modify the MAFSC cells (ortheir derivatives) can be found, for example, in standard textbooks andreviews in cell biology, tissue culture, and embryology. Methods inmolecular genetics and genetic engineering are described, for example,in Molecular Cloning: A Laboratory Manual, 2nd Ed. (Sambrook et al.,1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal CellCulture (R. I. Freshney, ed., 1987); the series Methods in Enzymology(Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells (I. M.Miller & M. P. Calos, eds., 1987); Current Protocols in MolecularBiology and Short Protocols in Molecular Biology, 3rd Edition (F. M.Ausubel et al., eds., 1987 & 1995); and Recombinant DNA Methodology II(R. Wu ed., Academic Press 1995).

Transduction of MAFSC cells can be accomplished by each of manytechniques, DNA or RNA gene/sequence insertion of a suitably promotedgene construct, electroporation of said genes, infection by retroviral,lentiviral or other viral vector constructs encoding a gene of interest,mechanical gene introduction or the transfer by any means of specificprotein, glycoprotein or phosphoprotein entities by any of a number ofgeneral techniques used for such purpose. The nucleic acid molecule ofinterest can be stably integrated into the genome of the host MAFSCcell, or the nucleic acid molecule and can also be present as anextrachromosomal molecule, such as a vector or plasmid. Such anextrachromosomal molecule can be auto-replicating. The term“transfection,” as used herein, refers to a process for introducingheterologous nucleic acid into the host MAFSC or MAFSC-derived cell ortissue. A transfected MAFSC cell refers to a MAFSC cell into which aheterologous nucleic acid molecule has been introduced. One example of auseful genetic modification of a MAFSC cell was the insertion of the“TERT” gene (telomerase reverse transcriptase). MAFSC Cells that havehad the Tert gene transduced by retroviral gene transduction expressedthis gene to high levels and had acquired an immortal phenotype, i.e.they did not senesce after >200 population doublings. Further, geneticmodification of MAFSC cells can be used for purposes of propagation ofstable non-differentiated cells, for the purpose of accomplishing stableor transient MAFSC cell differentiation or for gene therapy purposes,such as the administration of a gene encoding a functional proteinproduct to an individual that lacks a functional copy of a gene ofinterest. If desired, MAFSC cells or their derivatives can also begenetically modified to inhibit the expression of certain genes, usinggene manipulation methods known in the art.

It will be understood by those of skill in the art that numerous andvarious modifications can be made without departing from the spirit ofthe present invention. A more complete understanding can be obtained byreference to the following specific examples which are provided hereinfor purposes of illustration only and are not intended to limit thescope of the invention.

EXAMPLES Example 1 Isolation and Expansion of Undifferentiated CellsDerived from Amniotic Fluid

Approximately 2 to 5 ml of fresh amniotic fluid was harvested from womenundergoing routine amniocentesis at 16 to 21 weeks of pregnancy (2^(nd)trimester). Second trimester amniotic fluid contained approximately1-2×10⁴ live cells per ml. The cells were pelleted in a clinicalcentrifuge and resuspended in 15 ml “MAFSC” medium. MAFSC medium wascomposed of low glucose Dulbecco Modified Eagle's Medium (GIBCO,Carlsbad, Calif.) and MCDB 201 medium (SIGMA, Saint Louis, Mo.) at a oneto one ratio and contained 2% Defined Fetal Calf Serum (HYCLONE, Logan,Utah), 1× insulin-transferrin-selenium,linoleic-acid-bovine-serum-albumin (ITS+1, SIGMA), 1 nanomolardexamethasone (Sigma), 100 μm ascorbic acid 2-phosphate (Sigma), 4 μm/mlgentamycin, 10 ng/ml of rhEGF (R&D Systems, Minneapolis, Minn.), 10ng/ml rrPDGF-BB (R&D) and 10 ng/ml rhFGF-basic (R&D). The wells of6-well culture dishes were prepared for cell plating by coating for onehour at room temperature with 2.5 ml of fibronectin (stock of 10 μgfibronectin/ml of sterile water) immediately prior to cell plating. Thefibronectin solution was removed prior to cell plating and the wellswere not washed after removal of the fibronectin solution. The cellswere then seeded in 2.5 ml of medium in each well.

The cells in MAFSC culture appeared under the inverted phase microscopeas large suspension cells that divided on average once every 4 days, butceased dividing 8-12 days after seeding. The growth medium of MAFSCcultures was changed with complete MAFSC medium every two days makingsure to not lose the suspended cells. After 8-10 days, small numbers ofadherent cells emerged which grew into large colonies of >10⁵ cells in14-15 days. On average, 0-1 adherent colonies grew out per 2×10⁴ livecells seeded. Hence, a sample of 5 ml of fresh amniotic fluid gave riseto 3-5 adherent cell colonies, resulting in a single colony/clone in themajority of the wells of 6-well cell culture clusters.

Cells were transferred to successively larger fibronectin-coatedflasks/vessels. To perform cell transfer, the cells were grown to asubconfluent state of approximately 40% confluence and were detachedwith 0.25% Trypsin-EDTA and replated at a 1:3 or 1:12 dilution under thesame culture conditions.

Example 2 Morphological Characterization of Cell Types

Cultured amniotic fluid-derived cells were tested for cell surface anddifferentiation markers and were karyotyped. These cells were found tobe immortal or near-immortal and were named Multipotent Amniotic FetalStem Cells (MAFSC).

All of >80 amniotic fluid sample harvests of 5 ml gave rise to at leastone adherent MAFSC colony and continuous culture. The majority of sampleharvests gave rise to 3-4 individual clones. Among the individualclones, different colonies/cultures had diverse colony morphologies, asshown in FIG. 1. Some cultures had a flat, epihelial morphology (FIG.1A). Others had a fibroblastic morphology (FIGS. 1B, 1C). Both theepithelioid and the fibroblastic classes of cultures senesced after ˜60population doublings (PD), yielding a maximum of 10¹⁸ cells, unless thecells were immortalized by the expression of the human TERT (telomerase)gene that maintained the length of the cells' telomeres. Indeed, mortalMAFSC cultures have been immortalized at low (PD 15-25) transfer numbersby infection with an amphotropic high titer retroviral vector expressingthe human TERT gene. MAFSC cultures immortalized with TERT have notsenesced after >220 population doublings. Thus, the TERT-modified MAFSCcultures were immortal, though only after genetic modification which maynot be the advantaged way to derive human stem cell strains.

About half of the amniotic fluid samples gave rise to MAFSCclones/cultures that behaved like immortal cell lines, as shown in FIGS.2A and 2B. These cultures grew vigorously, with a doubling time of 28hours. When confluent, the cells piled up in multilayered fashion andnumerous round, semi-detached cells grew on top of a swirling,non-contact-inhibited layer of cells. These aggressive culturesexpressed the telomerase gene/protein. The cells were cloneable intosingle cell clones and are non-senescing. These vigorously growing MAFSClines expressed very high levels of a set of cell surface determinantsknown to be present on non-differentiated human Embryo Stem Cells (hES)and expressed a set of surface determinants known to be associated withnon-differentiated human Mesenchymal Stem Cells (MSC). MAFSC cells didnot express markers characteristic of hematopoietic cells, e.g. CD45 andCD34, see FIGS. 3 and 4, which show flow cytometry examples of one suchvigorous MAFSC line, #111a.

Example 3 FACS Analysis of MAFSC Cells

Cells were prepared for FACS analysis by trypsinizing to remove themfrom the tissue culture flask, washing in buffer, HBSS, 2% BSA, 0.1%sodium azide, then resuspended in 100 μL of the same buffer. Forintracellular antigens (i.e. Oct-4), the cells were fixed andpermeablized using Beckman-Coulter IntraPrep reagents, as suggested bythe manufacturer. Primary antibodies specific for the indicatedcell-surface or intracellular marker were added at a 1:10 dilution andincubated for 30 minutes at room temperature, then washed. For samplesusing primary antibodies that were not fluorescently-conjugated, thecells were then resuspended in 250 μL of buffer and the appropriatefluorescent-labeled secondary antibody was added at a 1:250 dilution andincubated for 30 minutes at room temperature. Labeled cells were washedand resuspended in buffer or 1% paraformaldehyde for analysis by aFACSCalibur flow cytometer. The data obtained from this analysis wereplotted as the x-axis being the number of cells analyzed per point andthe y-axis indicating the logarithm of fluorescent intensity of theantibody-labeled cells. The fluorescence was compared to control cellsthat were not labeled with antibody, to discount any backgroundfluorescence. The percent indicated was the fraction of cells that werepositive for the specific antibody-labeled antigen. The level ofantibody label (X-axis) is proportional to the concentration of thespecific antigen present on the cells.

Example 4 Stem Cell Markers on MAFSC Cells

MAFSC lines expressed very high levels of a set of cell surfacedeterminants known to be present on non-differentiated human Embryo StemCells (hES) and expressed a set of surface determinants known to beassociated with non-differentiated human Mesenchymal Stem Cells (MSC).MAFSC cells did not express markers characteristic of hematopoieticcells, e.g. CD45 and CD34, see FIGS. 3 and 4, which show flow cytometryexamples of one such vigorous MAFSC line, #111a. The flow cytometry wasperformed as described above in Example 3.

Mass cultures of the MAFSC cells strain 111a were characterized by veryhigh expression of the globoseries glycolipid antigens SSEA3 (96%),SSEA4 (96%), the lack of expression of a lactoseries oligosaccharideantigen, SSEA1, the expression of the keratin sulphate-related antigensTra-1-60 (71%) and Tra-1-81 (82%) and the tissue non-specific alkalinephosphatase-related antigen Tra-2-54 (63%), FIG. 3. The expression (orlack of expression, SSEA1) of these antigens is expressly exhibited bypluropotential, undifferentiated human embryo stem cells in which theexpression of these antigens is lost (or gained, SSEA1) by the inductionof differentiation with retinoic acid (Draper J S, Pigott C, Thompson JA, Andrews P W. 2002 Journal of Anatomy 200: 249-258). MAFSC cellsexpressed high levels of HLA Class I but not of HLA Class II, low levelsof CD 117 (c-kit ligand) and Stro-1, FIG. 3.

In addition to the embryo stem cell markers shown in FIG. 3 anddiscussed in Table 1, MAFSC cells (such as, for example the MAFSC 111 aline), expressed high levels of the antigen CD13 (99.6%) aminopeptidaseN, CD44 (99.7%) hyaluronic acid-binding receptor, CD49b (99.8%)collagen/laminin-binding integrin alpha2, and CD105 (97%) endoglin. Thisset of cell surface antigens is found on human mesenchymal stem cellsbut not normally on human embryo stem cells (M F Pittinger et al.,Science 284:143-147, 1999; S Gronthos et al., J Cell Physiol. 189:54-63,2001). Hence, the amniotic fluid-derived MAFSC cells, grown andpropagated as described here, represent a novel class of human stemcells that combined the characteristics of hES cells and of hMSC cellsand can be expected to differentiate into many diverse directions.

The amniotic fluid-derived stem cells also expressed the transcriptionfactor OCT-4. The human embryonic stem cell markers typically found onMAFSC cells are shown in Table 1 and are data obtained for MAFSC cells,clone 111a, cultured for >40 population doublings in MAFSC culturemedium. The markers typically displayed by long term MAFSC cells arecompared with the same markers found on fresh amniotic fluid-derivedcells, “AES”, and with various control cells, the human embryoniccarcinoma cell line NTERA-4; amniotic-fluid-derived, long-termfibroblasts, MY-TERT, immortalized by the human telomerase reversetranscriptase gene, TERT; and normal human foreskin fibroblasts, HFF.Expression of the various markers is further described in co-pendingU.S. Patent Application filed Aug. 13, 2004, entitled, “MultipotentAmniotic Fetal Stem Cells.” TABLE 1 Embryonic Stem Cell Markers NTERA-4Embryonic MY-Tert HFF Marker MAFSC 111a “AES” Carcinoma Immortal Amnio-Foreskin (% Positive) Cultured Amniocytes Fresh Amniocytes Linefibroblasts Fibroblasts SSEA-1 3.3 n.d.* 3.8 2 1.5 SSEA-3 96 19.4 9.117.3 2.3 SSEA-4 96 39.8 96.8 67.8 8.2 Tra-1-60 71 48.9 99.8 4.9 1.7Tra-1-81 82 42   99.6 2.8 1.7 Tra-2-54 63 30.4 99.2 16.1 27.9 Oct-4 3912.7 99 2.6 3.4*n.d. = not determined

Example 5 Cryopreservation and Banking of Fresh Amniocentesis-Derivedand of Cultured MAFSC Cells

Both fresh amniocentesis-derived cells and cultured MAFSC cells werecryopreserved for banking purposes. Samples of amniotic fluid rangingfrom 2 to 5 ml were harvested. The cells were centrifuged to removeexcess amniotic fluid. The cells were then frozen in medium containing10% dimethyl sulfoxide and 25% fresh, filtered (0.10 micron) amnioticfluid (DMSO/AF freezing medium). Alternatively, the cells were grown toproduce MAFSC cultures, which were then frozen as above. The freshamniocentesis-derived cells and cultured MAFSC cells were frozen inDMSO/AF freezing medium in a controlled-rate liquid nitrogen freezer at1° C./min. Frozen samples were stored under liquid nitrogen in freezingampoules.

Example 6 Establishment of a Universal Stem Cell Bank Composed ofTransplantable Amniocentesis-Derived Stem Cells

To assure the availability of stem cells transplantable into all or intoa majority of potential recipients, stem cells will be banked thatpossess a range of genetic characteristics and that display a range ofantigens, for example a range of human leukocyte antigens (HLAantigens). Following widely recognized and universally appliedtransplantation methodologies in human bone marrow transplantation andin human organ transplantation, matching of ten HLA specificities isgenerally sufficient to achieve full and problem-free transplantations.The development of an amniocyte-derived stem cell bank facilitates thecryopreservation of hundreds or of thousands of lines possessingfinely-mapped transplantation specificities for the accomplishment ofroutine stem cell transplantation into a majority of potential subjects.

Example 7 Linkage of Cell “Bank” to Searchable Database

The cryopreserved cells may be listed in a searchable database.Information such as genetic background of the stored cells, familialdata, date of submission, suspected or known genetic diseases ofindividuals or relatives, etc., may be included, for example. When anindividual has a medical problem that can be alleviated or reversed byadministering cells derived from the thawed cryopreserved cells, thedatabase can be searched for pertinent information. A suitable samplecan then be chosen for thawing, cell proliferation, differentiation, andadministration to the individual.

Example 8 Cell Recovery Procedures and Viability of Thawed Cells

Cell thawing was done rapidly in a 37° C. water bath resulting in therecovery of >99% of the frozen AF and MAFSC cells. Thawed cells werethen grown by being immediately diluted 10-fold to reduce theconcentration of DMSO to <1%. The percent viability of the cells wasdetermined. When the amniotic fluid cells are properly frozen, as is thestate of the art, and they are rapidly thawed, there is essentially nocell death: viability is close to 99%, the cells' growth behavior inMAFSC cultures is also not affected by cryopreservation and thawing.

Example 9 Measurements of the Viability of the Cryopreserved Cells

The viability of cryopreserved cells was equal to that of fresh amnioticfluid cells. A typical fresh 2nd trimester amniocentesis sample had aviablility of 30-50%, depending on the exact number of weeks of thepregnancy: earlier harvests were more highly viable. At later times inthe pregnancy, e.g. 35 weeks, the viability of freshly removed amnioticcells is about 10%, however the absolute number of viable cells in asample is roughly equal to that found in 16-18 week amniocentesisharvests, namely 1-2×10⁴ cells/ml. At later times in the pregnancy moredead cells accumulate, while the absolute number of live cells stayssimilar.

The time between the harvesting of the amniotic cells to the time ofseeding the cells in MAFSC culture medium or to the time ofcryopreserving the cells was important. A delay of 24 hours inseeding/growing or in cryopreserving the cells reduced the probabilityof success in growing MAFSC stem cells by ˜90%. Hence, speedycryopreservation of amniotic fluid samples, within 2-4 hours ofamniocentesis drawing, was crucial in the success of growing MAFSC stemcells from the sample. Interestingly, the concentration of viable cellsdid not diminish significantly by delaying the amniotic fluid sampleprocedures (seeding or freezing) by keeping the samples at roomtemperature in their natural fluid. The number of resultant stem cells,however, was significantly reduced, by ˜90%. Therefore, the MAFSC stemcells in an amniotic fluid sample were more fragile than the bulk of thecells in the harvest.

Example 10 Use of Revived Cells to Differentiate into Various Cell Types

Cryopreserved cells were revived, cultured, and differentiated intovarious cell types, such as neural cells, adipogenic cells, andchondrogenic cells, as described in co-pending U.S. Patent Applicationfiled Aug. 13, 2004, entitled, “Multipotent Amniotic Fetal Stem Cells,”which is incorporated by reference herein in its entirety.

Example 11

Treatment of Diseases Using Transplantation of Differentiated MAFSCs

Cryopreserved MAFSCs that have been thawed from a cell bank system orMAFSC stem cells that have been grown from a cryopreserved amnioticfluid cell sample may be differentiated into various tissues, as needed.In one example, MAFSC stem cells can be differentiated into pancreaticbeta cells that secrete insulin under the control of glucose prevalence.Beta cells or beta cells enveloped into pancreatic islets with orwithout scaffolding can be implanted at a suitable site in diabeticpatients. In this way, the glucose responsive beta cells orreconstructed pancreatic islets can control the level of glucose in thediabetic patient. The availability of syngenetic (“own”) amniotic cellsor the genetic matching of amniotic fluid-derived islet cells to adiabetic's transplantation antigen status thus facilitates theregulation of glucose concentration without the danger ofersatz-pancreatic rejection by the diabetic recipient.

Example 12 Use of Thawed MAFSCs to Promote Bone Marrow Regeneration

Following MAFSC cell differentiation into the cells of interest,differentiated MAFSCs can be transplanted into a patient in need oftreatment to promote bone marrow regeneration. MAFSC cells of suitabletransplantation genotype, either the fetal-donor of origin of thecryopreserved amniotic cells or a transplantation antigen-matchedrecipient patient, are differentiated in vitro by state of the artmethods into hematopoietic stem and/or progenitor cells, for example bymethods similar to those described by Carotta et al., “Directeddifferentiation and mass cultivation of pure erythroid progenitors frommouse embryonic stem cells”, Blood 2004, May 27, prepublished online,DOI 10.1182. MAFSC cells differentiated into specific hematopoieticstem/progenitor cell types can then be used for transplantation byinfusion into recipients in need of hematopoietic cell transplantation.The cryopreservation of millions of samples of amniotic fluid cells andthe potential generation of multiple MAFSC stem cell lines of differenttransplantation specificities facilitates the preparation, bydifferentiation, of suitable hematopoietic stem/progenitor cells as ageneral bone marrow-transplantation resource. Genetically andantigenically matched hematopoietic stem/progenitor cell populationswill induce minimal transplantation complications in the transplantationof patients requiring hematopoietic cell transplantation.

Example 13 Genetic Modification of MAFSCs to Express the TERT Gene toAlter MAFSC Properties

MAFSCs or MAFSC-derived cells may also be genetically modified eitherbefore or after the banking period, using any suitable means, such ascell transfection procedures. The nucleic acid molecule can be stablyintegrated into the genome of the host MAFSC cell, or the nucleic acidmolecule and can also be present as an extrachromosomal molecule, suchas a vector or plasmid. One example of a useful genetic modification ofa MAFSC cell is the insertion of the “TERT” gene (human telomerasereverse transcriptase, GenBank Accession No. NM_(—)003219). A vectorsuitable for mammalian transfection is prepared, containing a selectablemarker gene and the gene encoding “TERT”, operably linked to a suitablepromoter sequence. The vector is used to stably transfect a mammalianMAFSC cell. Stably transfected cells are then selected using theselection agent corresponding to the selectable marker gene. Expressionof TERT is then confirmed using antibody-based detection procedures.MAFSC Cells that are able to express this TERT sequence can besignificantly expanded.

Example 14 Use of an Amniotic Fluid-Derived Cell Cell Banking andRecording System to Find Samples of Similar Type for Matching Purposes

An individual in need of stem cells or stem cell products for treatmentof a disease is identified. Type identifiers of the individual areidentified. The database of amniotic fluid-derived cell banked samplesis searched for donors with similar type identifiers. Further analysisis performed to confirm the similarity. The amniotic fluid-derived cellsample is retrieved, and the cells are revived. The cells are treated soas to expand and differentiate into the desired cell type, and aresubsequently transplanted to the individual in need of treatment. Thesuccess of the treatment is ascertained at daily to monthly intervals.Using this method, the disease is successfully treated.

It will be appreciated that no matter how detailed the foregoing appearsin text, the invention can be practiced in many ways. As is also statedabove, it should further be noted that the use of particular terminologywhen describing certain features or aspects of the present inventionshould not be taken to imply that the broadest reasonable meaning ofsuch terminology is not intended, or that the terminology is beingre-defined herein to be restricted to including any specificcharacteristics of the features or aspects of the invention with whichthat terminology is associated. Thus, although this invention has beendescribed in terms of certain preferred embodiments, other embodimentswhich will be apparent to those of ordinary skill in the art in view ofthe disclosure herein are also within the scope of this invention.Accordingly, the scope of the invention is intended to be defined onlyby reference to the appended claims and any equivalents thereof. Alldocuments cited herein are incorporated herein by reference in theirentireties.

1. A cell bank system, comprising: a plurality of preserved, viablesamples, containing amniotic fluid-derived cells, wherein said samplesare from a plurality of individuals; and a database containing one ormore data fields that allow for specific identification and retrieval ofindividual samples.
 2. The cell bank system of claim 1, wherein saiddatabase includes data fields allowing association of an individualsample with a person from whom the sample was obtained.
 3. The cell banksystem of claim 1, wherein the samples are cryopreserved.
 4. The cellbank system of claim 1, wherein individual samples are retrievable bythe person from whom the sample was obtained.
 5. The cell bank system ofclaim 1, wherein said samples contain MAFCS.
 6. The cell bank system ofclaim 1, wherein said samples contain amniotic fluid.
 7. The cell banksystem of claim 1, wherein said samples contain multipotent orpluripotent stem cells.
 8. A method for banking stem cells, comprising:obtaining a plurality of viable samples containingamniotic-fluid-derived stem cells, wherein said samples are from theamniotic fluid of a plurality of individual fetuses; preserving andstoring the samples in a manner that preserves viability of at leastsome of said stem cells; storing data relating to the identity ofindividual samples; and providing for retrieval of said individualsamples by or on behalf of the individual from whom the sample wasobtained.
 9. The method of claim 8, wherein the samples arecryopreserved.
 10. The method of claim 8, wherein said samples containMAFCS.
 11. The method of claim 8, wherein said samples contain amnioticfluid.
 12. The method of claim 11, wherein said amniotic fluid comprisesabout 15% to 35% (V/V) of said medium.
 13. The method of claim 8,wherein said samples contain multipotent or pluripotent stem cells. 14.A collection comprising a plurality of viable stem cells derived fromamniotic fluid of different fetuses.
 15. The collection of claim 14,wherein said stem cells are cryopreserved.
 16. The collection of claim14, wherein said cells are fresh cells isolated from amniotic fluid. 17.The collection of claim 14, wherein said cells are cultured MAFSC cells.18. A method of preparing amniotic fluid derived stem cells (MAFSC) fora potential future use, comprising: obtaining amniotic fluid containinglive cells; culturing MAFSC cells isolated from said amniotic fluid; andcryopreserving said MAFSC cells.
 19. A storage bank of multiple samplesof amniotic fluid-derived cells or MAFSCs taken from multipleindividuals, wherein the samples are preserved in such a way as to beviable upon recovery.
 20. The storage bank of claim 19, wherein thecells are cryopreserved in a medium comprising DMSO.
 21. The storagebank of claim 19, wherein at least some of the samples aredifferentiable human stem cells.
 22. The storage bank of claim 21,wherein said human stem cells are multipotent.
 23. The storage bank ofclaim 21, wherein said human stem cells are pluripotent.
 24. The cellbank system of claim 1, wherein said amniotic fluid-derived cellscomprise pluripotent stem cells.
 25. The cell bank system of claim 1,wherein said amniotic fluid-derived cells comprise multipotent stemcells characterized by a) the ability to grow in continuous culture forat least 60 generations, and b) the presence of at least one markerselected from the group consisting of: SSEA3, SSEA4, Tra1-60, Tra1-81,Tra2-54, and Oct-4.
 26. The cell bank system of claim 1, wherein saidamniotic fluid-derived cells comprise multipotent stem cellscharacterized by a) the ability to grow in continuous culture for atleast 60 generations, and b) the presence of all of the markers SSEA3,SSEA4, Tra1-60, Tra1-81, Tra2-54, and Oct-4.
 27. The cell bank system ofclaim 25 wherein said stem cells are further characterized by expressingat least one marker selected from the group consisting of: HLA Class I,CD13, CD44, CD49b, and CD105.
 28. The cell bank system of claim 1,wherein said amniotic fluid-derived cells comprise pluripotent stemcells characterized by a) the ability to grow in continuous culture forat least 60 generations, and b) the presence of at least one of themarkers selected from the group consisting of: SSEA3, SSEA4, Tra1-60,Tra1-81, Tra2-54, and Oct-4.
 29. The cell bank system of claim 1,wherein said amniotic fluid-derived cells comprise pluripotent stemcells characterized by a) the ability to grow in continuous culture forat least 60 generations, and b) the presence of the following markers:SSEA3, SSEA4, Tra1-60, Tra1-81, Tra2-54, and Oct-4.
 30. The cell banksystem of claim 29 wherein said stem cells are further characterized byexpressing at least one marker selected from the group consisting of:HLA Class I, CD13, CD44, CD49b, and CD105.